One of the most riveting detective stories of the last century
supposedly ended in 1998, when the Russian government declared that
bones excavated from a Siberian mass grave seven years earlier
indeed belonged to the Romanovs, Russia's last royal family, who
were executed by the Bolsheviks in 1918.

A new study, however, is reopening the book.

The Romanovs, Russia’s last royal
family, were executed by the Bolsheviks in 1918. A team led by
Stanford senior scientist Alec Knight has cast doubt on a 1994
report that claims to have identified the family’s
remains.Courtesy of Hoover
Institution

A team led by Alec Knight, a senior scientist in the Stanford
lab of anthropological sciences Assistant Professor Joanna
Mountain, argues that previous DNA analyses of the purported
Romanov remains -- nine skeletons unearthed near Ekaterinburg in
central Russia -- are invalid. Knight and his colleagues base their
claim on molecular and forensic inconsistencies they see in the
original genetic tests, as well as their independent DNA analysis
of the preserved finger of the late Grand Duchess Elisabeth --
sister of Tsarina Alexandra, one of the 1918 victims -- which
failed to match the tsarina's own DNA. The Stanford team's findings
are reported in the January/February issue of the Annals of
Human Biology.

Flawed experiments?

The original DNA analysis was arranged by the Russian
government's Commission on the Identification of the Remains. As
reported in Nature Genetics in 1994, Peter Gill of Britain's
Forensic Science Service and Pavel Ivanov, a Russian geneticist
from the Engelhardt Institute in Moscow, conducted a battery of
experiments supporting the hypothesis that the Ekaterinburg bones
belonged to the Romanovs. The team performed DNA-based sex testing
and analyzed short sequences of DNA from the nucleus of bone cells
to establish that the remains of the alleged tsar, tsarina and
three daughters belonged to the same family.

To solidify these conclusions, Gill and Pavel also examined DNA
from mitochondria, the energy-producing organelles within cells.
Compared to DNA found inside the nucleus, mitochondrial DNA
preserves well in bones and acquires mutations very slowly --
making it a prized specimen for multigenerational forensic
analysis. But there is a catch: Mitochondrial DNA is passed only
along the maternal line. For the 1994 study, researchers determined
the sequence of mitochondrial DNA fragments from the presumed
Romanov skeletons and found that they matched DNA sequences
obtained from blood samples of Britain's Prince Philip (Tsarina
Alexandra's grandnephew) and two living relatives of the tsar's
maternal grandmother.

Knight argues these results are too good to be true. In
particular, he doubts the researchers could have obtained such long
stretches of DNA sequence (a string of 1,223 bases, DNA's chemical
building blocks) from old bones. Citing standards for ancient DNA
analysis that were established several years after the 1994
publication, Knight contends that DNA from skeletal remains that
spent over 70 years in a shallow, earthen grave would have degraded
so severely that sequences longer than 250 bases would have been
nearly impossible to recover in lab experiments.

"Based on what we know now, those bones were contaminated,"
Knight said. He considers the successful amplification of a
1,223-base sequence from all nine skeletons in the original study
as "certain evidence" that the bone samples were tarnished with
fresh, less-degraded DNA -- perhaps from an individual who handled
the samples.

As reported recently in Science, Gill maintains that his
team's DNA analysis "set the standard." He says that Knight's paper
mischaracterizes his work and "comes across as vindictive and
political."

Blood-soaked evidence

Peter de Knijff, head of the Forensic Laboratory for DNA
Research at Leiden University Medical Center in the Netherlands,
agrees with Knight's assessment that the Gill-Ivanov study was
"unrealistically solid."

De Knijff's qualms about the original study also stem from
Ivanov's unwillingness to disclose results from his analysis of a
blood-soaked handkerchief that Tsar Nicholas II used to treat a
head wound suffered after he was struck by a would-be assassin in
Japan in 1891. The handkerchief is a "potentially pristine source
of DNA of the last tsar," according to de Knijff, noting that
Ivanov refused to disclose experimental details and claimed that
the handkerchief DNA was degraded and hence analysis was unfeasible
-- an assertion that other scientists dispute.

These sorts of irregularities provided the original impetus for
Knight's entry into the forensic debate. About three years ago,
Daryl Litwin, an author of the Knight et al. paper who was studying
law in Sacramento, proposed to Knight the idea for a re-analysis of
the Gill-Ivanov data after reading Robert Massie's book The
Romanovs: The Final Chapter. "I just kept finding contradiction
and discrepancy from point to point," Litwin said. "I was left kind
of befuddled." Before approaching Knight, Litwin discussed his
ideas with a Russian history expert at the Hoover Institution at
Stanford, who agreed that the Romanov verdict was worth
re-examining.

Months later, Knight went to New York to procure a small wooden
case containing a finger of Grand Duchess Elisabeth. Since the 1982
opening of Elisabeth's coffin in Jerusalem, the finger had been
preserved in a reliquary at the New York home of Bishop Anthony
Grabbe, president of the now-disbanded Orthodox Palestine
Society.

Though Knight's trip was funded by the Russian Expert Commission
Abroad -- a group of about 20 scholars in the West and Russia who
challenge the assertion that the bones are royal -- Knight
maintains that his experiments were unbiased. "[The Commission
Abroad] didn't do the science," he said. "They just bought me the
plane ticket and got me the sample. They had no control over the
work."

Continuing controversy

Nevertheless, some scientists -- several of whom participated in
the original DNA analyses -- are unconvinced by Knight's
conclusions. Mark Stoneking, a molecular anthropologist at the Max
Planck Institute for Evolutionary Anthropology in Leipzig, Germany,
concludes that "it is certainly plausible that DNA preservation was
sufficient to permit amplification of large fragments."

Tom Parsons of the Armed Forces DNA Identification Laboratory in
Rockville, Md., and Victor Weedn, a forensic scientist who
established the U.S. military's DNA identification program, agree
that the discovery of the Ekaterinburg remains in an area of
permafrost explains how larger DNA fragments were stable enough to
be recovered in the original analyses.

Knight agrees that frozen DNA is more stable but points out that
Ekaterinburg -- which is at the same latitude as Moscow and just
north of Kazakhstan -- can reach 100 degrees Fahrenheit in July and
August.

Meanwhile, as scientists squabble over finer details of the
forensic analysis, historians seem content with a more holistic
view of the Romanov drama.

"There may be some ambiguity about which physical remains belong
to whom, but this uncertainty doesn't really change our fundamental
understanding of the Russian Revolution and the nature of
Bolshevism," said Robert Crews, an assistant professor of history
at Stanford.

Donald Ostrowski, a Russian historian at Harvard University,
said he had doubts about the Ivanov and Gill analysis of the bones,
so he "just decided to eliminate it from [his] consideration of the
historical evidence." Though he has concluded from probabilistic
analysis of existing evidence that the bones belong to the
Romanovs, Ostrowski remains open to hearing new evidence or
re-analysis of old data. "The case is by no means closed," he
said.